The objective of this project is to design and develop a locally acting, allosteric inhibitor of the enzyme transglutaminase 2 (TG2). TG2 activity is central to the pathogenesis of celiac sprue, a gluten induced autoimmune disease of the small intestine for which no pharmacotherapy exists. Toward this end two specific aims are proposed in this application: 1) Characterize TG2 inhibitors using a non-denaturing polyacrylamide gel electrophoresis (PAGE) assay which distinguishes the closed, inactivated conformation of TG2 from the open, activated form. These studies are designed to distinguish allosteric inhibitors of the inactivated state from active site inhibitors. 2) Design, synthesize and assay new allosteric inhibitors which have been engineered for improved potency. This research is intended to identify one or more lead series for optimization in subsequent work. Initial work will involve testing TG2 inhibitors identified in Preliminary Studies with the non-denaturing PAGE assay. The results of these experiments will be used to refine a predictive in silico model for allosteric TG2 inhibitors. Next, a large and diverse virtual library of potential allosteric TG2 inhibitors will be defined, encompassing approximately 3B compounds. Compounds from this space will be sampled and optimized using a closed-loop engineering process in which the predictive model for TG2 inhibitors will serve as the primary objective function. A small set (6-9) of the most promising engineered inhibitors will be synthesized and characterized in biochemical, cellular and conformational assays of TG2 inhibition. The projected outcome of these experiments is to identify and qualify a lead series for further optimization. PUBLIC HEALTH RELEVANCE: This research involves the design of a medicine to treat celiac sprue, an autoimmune disease of the small intestine. No therapeutic agent is currently available to treat celiac sprue, which afflicts 0.5-1% of the population.